PsiepsilonRACK peptide composition and method for protection against tissue damage due to ischemia

ABSTRACT

A method of reducing damage to cells and tissue caused by an ischemic or hypoxic event is disclosed. The method includes administering to the cell or tissue, either in vivo or ex vivo, ψεRACK peptide. The peptide can be administered before, during or after the ischemic or hypoxic event.

[0001] This application is a continuation of U.S. application Ser. No.10/007,363 filed Nov. 9, 2001, now pending; which claims the benefit ofU.S. Provisional Application No. 60/247,830 filed Nov. 10, 2000, both ofwhich are incorporated herein by reference in their entirety.

[0002] This work was supported in part by The National Institutes ofHealth Grant HL52141. Accordingly, the United States Government hascertain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to a method of protecting cells andtissues from damage due to an ischemic event. The method involvesadministering a peptide agonist of protein kinase C, and morespecifically, administering a pseudo-epsilon RACK (ψεRACK) peptide.

REFERENCES

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BACKGROUND OF THE INVENTION

[0038] Protein kinase C (PKC) is a key enzyme in signal transductioninvolved in a variety of cellular functions, including cell growth,regulation of gene expression and ion channel activity. The PKC familyof isozymes includes at least 11 different protein kinases which can bedivided into at least three subfamilies based on their homology andsensitivity to activators. Members of the classical or cPKC subfamily,α, β_(I), β_(II) and γPKC, contain four homologous domains (C1, C2, C3and C4) inter-spaced with isozyme-unique (variable or V) regions, andrequire calcium, phosphatidylserine (PS), and diacylglycerol (DG) orphorbol esters for activation. Members of the novel or nPKC subfamily,δ, ε, η, and θPKC, lack the C2 homologous domain and do not requirecalcium for activation. Finally, members of the atypical or αPKCsubfamily, ζ and λ/ιPKC, lack both the C2 and one half of the C1homologous domains and are insensitive to DG, phorbol esters andcalcium.

[0039] Studies on the subcellular distribution of PKC isozymesdemonstrate that activation of PKC results in its redistribution in thecells (also termed translocation), such that activated PKC isozymesassociate with the plasma membrane, cytoskeletal elements, nuclei, andother subcellular compartments (Saito, et al., 1989; Papadopoulos andHall, 1989; Mochly-Rosen, et al., 1990).

[0040] It appears that the unique cellular functions of different PKCisozymes are determined by their subcellular location. For example,activated β_(I)PKC is found inside the nucleus, whereas activatedβ_(II)PKC is found at the perinucleus and cell periphery of cardiacmyocytes (Disatnik, et al., 1994). Further, in the same cells, εPKCbinds to cross-striated structures (possibly the contractile elements)and cell-cell contacts following activation or after addition ofexogenous activated εPKC to fixed cells (Mochly-Rosen, et al., 1990;Disatnik, et al., 1994). The localization of different PKC isozymes todifferent areas of the cell in turn appears due to binding of theactivated isozymes to specific anchoring molecules termed Receptors forActivated C-Kinase (RACKs).

[0041] RACKs are thought to function by selectively anchoring activatedPKC isozymes to their respective subcellular sites. RACKs bind onlyfully activated PKC, but RACKs are not necessarily substrates of theenzyme nor is the binding to RACKs mediated via the catalytic domain ofthe kinase (Mochly-Rosen, et al., 1991). Translocation of PKC reflectsbinding of the activated enzyme to RACKs anchored to the cellparticulate fraction and the binding to RACKs is required for PKC toproduce its cellular responses (Mochly-Rosen, 1995). Inhibition of PKCbinding to RACKs in vivo inhibits PKC translocation and PKC-mediatedfunction (Johnson, et al., 1996; Ron, et al., 1995; Smith andMochly-Rosen, 1992).

[0042] cDNA clones encoding RACK1 and RACK2 have been identified (U.S.Pat. No. 5,519,003; Ron, et al., 1994; Csukai, et al., 1995). Both arehomologs of the β subunit of G proteins, a receptor for anothertranslocating protein kinase, the β-adrenergic receptor kinase, βARK(Pitcher, et al., 1992). Similar to Gβ, RACK1, and RACK2 have seven WD40repeats (Ron, et al., 1994; Csukai, et al., 1995). Recent data suggestthat RACK1 is a β_(II)PKC-specific RACK (Stebbins et al., 2001) whereasRACK2 is specific for activated εPKC (Csukai et al., 1997).

[0043] Translocation of PKC is required for proper function of PKCisozymes. Peptides that mimic either the PKC-binding site on RACKs(Mochly-Rosen, 1991a; Mochly-Rosen, 1995) or the RACK-binding site onPKC (Ron, et al., 1995; Johnson, et al., 1996) are isozyme-specifictranslocation inhibitors of PKC that selectively inhibit the function ofthe enzyme in vivo. For example, an eight amino acid peptide derivedfrom εPKC (peptide εV1-2; SEQ ID NO:1, Glu Ala Val Ser Leu Lys Pro Thr)is described in U.S. Pat. No. 6,165,977. The peptide contains a part ofthe RACK-binding site on εPKC and selectively inhibits specificεPKC-mediated functions in cardiac myocytes.

[0044] Recently, PKC and more specifically εPCK have been shown to beinvolved in cardiac preconditioning to provide protection from ischemicinjury. Prolonged ischemia causes irreversible myocardium damageprimarily due to death of cells at the infarct site. Studies in animalmodels, isolated heart preparations and isolated cardiac myocytes inculture have demonstrated that short bouts of ischemia of cardiac musclereduce such tissue damage in subsequent prolonged ischemia (Liu, Y., etal., 1995, 1996; Hu, et al., 1995; Brew, et al., 1995; Schultz, et al.,1996). This protection, which occurs naturally following angina and hasbeen termed preconditioning, can be mimicked by a variety ofnon-specific PKC agonists (Mitchell et al., 1993; Mitchell et al., 1995;Murry et al., 1986; Speechly-Dick et al., 1994). Both δPKC and εPKCactivation occurs following preconditioning (Gray et al., 1997),however, εPKC activation is required for protection of cardiac myocytesfrom ischemia-induced cell death (U.S. Pat. No. 6,165,977).

[0045] In a recent study, an εPKC-selective peptide agonist was shown toprovide cardio-protection from ischemia when administered intracellularyto isolated neonatal and adult cardiomyocytes and when producedintracellulary in vivo in transgenic mice (Dorn G. et al., 1999). Inthis work, a εPKC peptide agonist was administered intracellulary toisolated cells in vitro by laboratory techniques suitable at thecellular level or by genetic transfection. Unfortunately, neither ofthese techniques are suitable or likely to be successful for humantherapy. Moreover, it is unknown from this work whether or not the εPKCpeptide could be delivered extracellulary to whole tissue or intactorgans in vivo to achieve a therapeutic effect.

SUMMARY OF THE INVENTION

[0046] Accordingly, it is an object of the invention to provide a methodof protecting tissue from damage due to an ischemic event.

[0047] It is a further object of the invention to provide a method ofadministering an εPKC peptide agonist for induction of ischemicpreconditioning.

[0048] It is yet another object of the invention to provide a method ofameliorating damage to tissue caused by an ischemic event.

[0049] Accordingly, in one aspect, the invention includes a method ofreducing injury to a cell exposed to an ischemic or an hypoxic conditionby administering to the cell a ψεRACK peptide. In one embodiment, thepeptide is administered prior to exposing the cell to the ischemic orhypoxic condition. For example, the peptide administered for a period oftime of between about 1-180 minutes prior to exposing the cell toischemia or hypoxia. In another embodiment, the peptide is administeredafter the cell is exposed to an ischemic or hypoxic condition. Forexample, the peptide is administered for between about 1-180 minutesafter the cell is exposed to an ischemic or hypoxic condition. Inanother embodiment, the peptide is administered during to the cellduring the period of ischemia or hypoxia.

[0050] In one embodiment, the ψεRACK peptide has a sequence identifiedas SEQ ID NO:2. In other embodiments, the peptide has a sequenceselected from the group consisting of SEQ ID NOS:6-18.

[0051] In yet another embodiment, the ψεRACK peptide is linked to amoiety effective to facilitate transport across a cell membrane, such asa Tat-derived peptide (SEQ ID NO:5), an Antennapedia carrier peptide(SEQ ID NO:3), or a polyarginine peptide.

[0052] The peptide can be administered by a route selected from thegroup consisting or intraveneous, parenteral, subcutaneous, inhalation,intranasal, sublingual, mucosal, and transdermal.

[0053] In another aspect, the invention includes a method of reducinginjury to tissue exposed to an ischemic or an hypoxic condition byadministering to the tissue a ψεRACK peptide, as described above.

[0054] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055]FIG. 1A shows creatine phosphokinase (CPK) release as a functionof time during reverse perfusion in ex vivo rat hearts treated withψεRACK (open circles) or with scrambled ψεRACK (control, open squares)prior to an ischemic event. The treated hearts were compared to heartssubjected to the ischemic period but left untreated (closed triangles)and to hearts maintained under normoxia conditions (no ischemia, nopeptide treatment; closed squares) as controls.

[0056]FIG. 1B shows the total amount of CPK released in the studydescribed in FIG. 1A during 30 minutes of reperfusion.

[0057]FIG. 2A shows the functional recovery of a working heart perfusedwith ψεRACK prior to global ischemia, where the left ventricle developedpressure (LVP, in mmHg), its first derivative (CLP/dt, in mmHg/sec), andthe coronary perfusion pressure (PP, in mmHg) are shown. The right panelshows an expansion of the same trace before (baseline) and afterreperfusion.

[0058]FIG. 2B is a scan similar to FIG. 2A for a working heart perfusedwith scrambled ψεRACK prior to ischemia.

[0059]FIG. 3A shows CPK release as a function of time following ischemicinsult in ex vivo rat hearts treated for the first 20 minutes ofreperfusion with ψεRACK (open circles) and in hearts left untreatedafter ischemia (solid triangles).

[0060]FIG. 3B is a bar graph showing the total CPK release during a 60minute reperfusion period following an ischemic insult to whole rathearts (i) treated ex vivo with ψεRACK for 20 minutes after the ischemicinsult, or (ii) left untreated.

[0061]FIGS. 4A-4B are computer generated photos of pig heart slicestaken from pigs five days after an in vivo treatment with ψεRACK (FIG.4A) or with scrambled ψεRACK peptide as a control (FIG. 4B) during theinitial 10 minutes of a 30 minute ischemic insult.

[0062]FIG. 4C is a bar graph showing the total infarct area as a percentof area at risk, measured in grams of cardiac tissue, for pigs treatedwith ψεRACK peptide or with scrambled ψεRACK peptide (control).

[0063]FIG. 5 is a graph showing the ejection fraction, as measured byleft ventricurogram in pigs at three time points: (1) before occlusionof left anterior descending artery by balloon catheter (beforeocclusion); (2) immediately after reperfusion with ψεRACK (post ψεRACK);and (3) before sacrifice five days later (5 days post), for animalstreated with ψεRACK (solid triangles) and for control animals treatedwith a scrambled peptide (open circles).

BRIEF DESCRIPTION OF THE SEQUENCES

[0064] SEQ ID NO:1 is a prior art (U.S. Pat. No. 6,165,977) εPKCoctapeptide. SEQ ID NO:2 is a ψεRACK octapeptide SEQ ID NO:3 is theDrosophila Antennapedia homeodomain-derived carrier peptide. SEQ ID NO:4is a scrambled ψεRACK octapeptide. SEQ ID NO:5 is a Tat-derived carrierpeptide. SEQ ID NO:6 is a modification of SEQ ID NO:2. SEQ ID NO:7 is amodification of SEQ ID NO:2. SEQ ID NO:8 is a modification of SEQ IDNO:2. SEQ ID NO:9 is a modification of SEQ ID NO:2. SEQ ID NO:10 is amodification of SEQ ID NO:2. SEQ ID NO:11 is a modification of SEQ IDNO:2. SEQ ID NO:12 is a modification of SEQ ID NO:2. SEQ ID NO:13 is amodification of SEQ ID NO:2. SEQ ID NO:14 is a modification of SEQ IDNO:2. SEQ ID NO:15 is a modification of SEQ ID NO:2. SEQ ID NO:16 is amodification of SEQ ID NO:2. SEQ ID NO:17 is a modification of SEQ IDNO:2. SEQ ID NO:18 is a fragment of SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION

[0065] I. Definitions

[0066] “Tissue” as used herein refers to a group of similarlyspecialized cells that perform a common function. Tissues compose theorgans and structural components of living organisms. As used herein,tissue is intended to include an organ composed of a given tissue and tothe cells, individually or collectively, that compose the tissue.

[0067] “Ischemia” or an “ischemic event” refers to an insufficientsupply of blood to a specific cell, tissue or organ. A consequence ofdecreased blood supply is an inadequate supply of oxygen to the organ ortissue (hypoxia). Prolonged hypoxia may result in injury to the affectedorgan or tissue.

[0068] “Anoxia” refers to a virtually complete absence of oxygen in theorgan or tissue, which, if prolonged, may result in death of the cell,organ or tissue.

[0069] “Hypoxia” or a “hypoxic condition” intends a condition underwhich a cell, organ or tissue receives an inadequate supply of oxygen.

[0070] “Ischemic injury” refers to cellular and/or molecular damage toan organ or tissue or cell as a result of a period of ischemia.

[0071] “Hypoxic injury” refers to damage to a cell, organ, or tissue dueto a period of inadequate oxygen supply.

[0072] “Reperfusion” refers to return of fluid flow into a tissue aftera period of no-flow or reduced flow. For example, in reperfusion of theheart, fluid or blood returns to the heart through a supply line, suchas the coronary arteries in vivo, after removal of an occlusion to thefluid or blood supply.

[0073] “Treating a disease” refers to administering a therapeuticsubstance effective to reduce the symptoms of the disease and/or lessenthe severity of the disease.

[0074] “Conservative amino acid substitutions” are substitutions whichdo not result in a significant change in the activity (e.g.,εPKC-agonist activity or ψεRACK-agonist activity) or tertiary structureof a selected polypeptide. Such substitutions typically involvereplacing a selected amino acid residue with a different residue havingsimilar physico-chemical properties. For example, substitution of Glufor Asp is considered a conservative substitution since both aresimilarly-sized negatively-charged amino acids. Groupings of amino acidsby physico-chemical properties are known to those of skill in the art.

[0075] With respect to a specific sequence, “conservative substitutionsthereof” refers to sequences that differ from the specific sequence byhaving conservative amino acid substitutions at one or more positions.

[0076] “Peptide” and “polypeptide” are used interchangeably herein andrefer to a compound made up of a chain of amino acid residues linked bypeptide bonds. Unless otherwise indicated, the sequence for peptides isgiven in the order from the amino termiums to the carboxyl terminus.

[0077] When a first peptide or polypeptide is said to “correspond” or tobe “homologous” to a second peptide or polypeptide fragment, it meansthat the peptide or fragments have a similarity in amino acid residuesif they have an alignment score of >5 (in standard deviation units)using the program ALIGN with the mutation gap matrix and a gap penaltyof 6 or greater (Dayhoff, M. O., in ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE (1972) Vol. 5, National Biomedical Research Foundation, pp.101-110, and Supplement 2 to this volume, pp. 1-10.) The two sequences(or parts thereof) are more preferably homologous if their amino acidsare greater than or equal to 50%, more preferably 70%, still morepreferably 80%, identical when optimally aligned using the ALIGN programmentioned above.

[0078] A polypeptide sequence or fragment is “derived” from anotherpolypeptide sequence or fragment when it has an identical sequence ofamino acid residues as a region of the another sequence or fragment.

[0079] An “εPKC agonist peptide” or “εPKC specific agonist peptide” isunderstood to mean a peptide between about 4 and about 30, preferablybetween about 5 and about 15, amino acids in length that is derived fromεPKC. In one embodiment, the εPKC agonist peptide is derived from theregion of εPKC between about amino acids 70 and 120, preferably betweenabout amino acids 80 and 100, more preferably between about amino acids85 and 92.

[0080] Abbreviations: “PKC”, protein kinase C; “RACK”, receptor foractivated C-kinase.

[0081] Abbreviations for amino acid residues are the standard 3-letterand/or 1-letter codes used in the art to refer to one of the 20 commonL-amino acids.

[0082] II. Administration of ψεRACK Peptide Agonist

[0083] In one aspect, the invention provides a method of protecting acell, a tissue, or an organ from damage due to an ischemic event or ahypoxic condition by administering a peptide capable of activatingsignaling proteins, such as PKC, that are activated in vivo by bindingto a cognate polypeptide such as a receptor protein (RACK). Regions ofhomology between the PKC signaling peptide and its RACK are termed“pseudo-RACK” sequences (ψ-RACK; Ron et al, 1994, 1995) and typicallyhave a sequence similar to the PKC-binding region of the correspondingRACK. A ψ-RACK sequence that acts as an εPKC specific agonist peptide isidentified herein as SEQ ID NO:2. This peptide, referred to herein asψεRACK, is an εPCK specific agonist peptide and induces translocation ofεPKC. Heretofore, studies have focussed on identifying drugs andpeptidomimetics that mimic the action of ψεRACK, as it was unknown ifthe peptide itself could be administered in vivo or ex vivo to a wholeorgan to induce translocation of εPCK to confer protection fromischemia. In studies performed in support of the invention, ψεRACK wasadministered ex vivo and in vivo to whole hearts prior to and afterexposure to an ischemic condition. The peptide reduced the extent ofischemic injury, as will now be described.

[0084] A. Administration of ψεRACK Peptide Agonist Prior to Ischemia

[0085] A peptide having the sequence identified herein as SEQ ID NO:2was synthesized and purified as described in the Methods section. Ascrambled ψεRACK peptide (SEQ ID NO:4) was also prepared to serve as acomparison or a negative control to the ψεRACK peptide. In some studies,the peptides were conjugated to a carrier peptide, such as DrosophilaAntennapedia homeodomain (SEQ ID NO:3). It will be appreciated thatadministration of the native peptide, that is the peptide unmodified byattachment to a carrier, is also contemplated. Carrier peptides otherthan Drosophila Antennapedia are also contemplated, and other exemplarycarrier peptides include Tat-derived peptide (SEQ ID NO:5, Fawell etal., 1994, Vives et al., 1997) or a polyarginine peptide (Mitchell etal., 2000; Rolhbard et al., 2000), or other like carries described inthe art (Lindgren et al., 2000; Schwarlze et al., 2000).

[0086] Animals were anesthetized as described in Example 1 and theirhearts were rapidly removed and cannulated for perfusion on aLangendorff apparatus. Hemodynamic parameters were monitored untilstabilized, typically for 10-20 minutes. After equilibration, ψεRACKpeptide (SEQ ID NO:2) or a scrambled ψεRACK peptide (SEQ ID NO:4) weredelivered to the hearts via the coronary arteries using 0.5 μM ofpeptide for 20 minutes.

[0087] To induce global ischemia, flow to the hearts was interrupted for45 minutes. The hearts were then reperfused for 30 minutes. Duringreperfusion, ischemia-induced cell damage was determined by measuringthe activity of creatine phosphokinase (absorbance at 520 nm) in theperfusate. Creatine phosphokinase is a cytosolic enzyme in cardiacmyocytes and its presence in the perfusate is proportional to the numberof cardiomyocytes damaged by the ischemia. The results are shown inFIGS. 1A-1B.

[0088]FIG. 1A shows the creatine phosphokinase (CPK) release as afunction of time during the 30 minute reperfusion of ex vivo heartstreated with 500 μM (0.5 μM) ψεRACK (open circles) or with 0.5 μMscrambled ψεRACK (open squares) prior to the ischemic event. Also shownis the CPK release for hearts subjected to the ischemic period butuntreated with a peptide (closed triangles) and to hearts maintainedunder normoxia conditions (no ischemia, no peptide treatment; closedsquares), as controls. The hearts treated with ψεRACK prior to ischemiahave a release of CPK close to the control hearts maintained undernormoxia conditions. In contrast, hearts treated with scrambled ψεRACKhave significant release of CPK, indicating significant cell damage.

[0089]FIG. 1B is a bar graph showing the total CPK release during thereperfusion period for the hearts treated with ψεRACK and with scrambledψεRACK. The total CPK release from hearts exposed to the ischemic eventbut left untreated are also shown.

[0090]FIGS. 1A-1B show that hearts treated with ψεRACK prior to anischemic event provides protection from damage resulting from asubsequent ischemic event. Accordingly, in one embodiment the inventioncontemplates a method of reducing or preventing injury to a tissueexposed to an ischemic or hypoxic event by administering to the tissuean amount of ψεRACK agonist peptide. The peptide can be administeredfrom between 1-180 minutes prior to the ischemic event, more preferablyfrom about 1-120 minutes prior to the ischemic event, more preferablyfrom about 1-60 minutes prior to the ischemic event. In anotherembodiment, a time period of no more than about 180 minutes, morepreferably no more than 120 minutes, still more preferably no more thanabout 60 minutes, lapses between cessation of peptide delivery and theischemic event.

[0091] In another study in support of the invention, the functionalrecovery of ex vivo hearts after an ischemic event was evaluated bymonitoring the isovolumic left ventricle pressure (LVP) at a constantpacing (3.3 Hz) and at a constant coronary flow (10 mL/min), asdescribed in Example 1. Prior to the ischemic event, the hearts weretreated with 500 nM ψεRACK (SEQ ID NO:2) or with scrambled ψεRACKpeptide (SEQ ID NO:4). After the 30 minute global ischemia, the heartswere monitored during a 30 minute reperfusion period. The results areshown in FIGS. 2A-2B.

[0092]FIG. 2A shows the results for the hearts treated with ψεRACKpeptide prior to global ischemia, and FIG. 2B shows the results forhearts treated with scrambled ψεRACK prior to ischemia. In comparing thebaseline levels and reperfusion levels of the treated and untreatedhearts, it is seen that administration of ψεRACK peptide before ischemiasignificantly reduced the ischemic injury. This is particularlyevidenced by comparing the recovery of left ventricle developed pressure(LVP) in the hearts pre-treated with ψεRACK to those pre-treated withthe scrambled peptide. A four-fold improvement in both the LVP recoveryand its first derivative (dP/dt) were achieved by pre-treating withψεRACK. Furthermore, ψεRACK reduced the elevated LVP end diastolicpressure and the coronary perfusion pressure (PP).

[0093] Accordingly, these studies (FIGS. 1 and 2) show that cellulardamage to a tissue due to ischemic or hypoxia is reduced byadministering ψεRACK prior to the ischemic event or the hypoxic event.In this study, the ψεRACK was administered through the coronary arteriesto the organ and for a period prior to exposure to the ischemic and/orhypoxic condition. The time period, as well as the dose of peptideadministered, can vary considerably, as will be discussed in more detailbelow.

[0094] B. Administration of ψεRACK Peptide Agonist Subsequent toIschemia

[0095] In another study performed in support of the invention, ψεRACKwas administered to hearts ex vivo after a prolonged ischemic period andwas effective to provide protection from ischemic injury. As describedin Example 2, whole rat hearts were perfused on a Langendorff apparatus.After a 30 minute equilibration period, global ischemia was induced bystopping fluid flow for 45 minutes. The hearts were then reperfused withor without ψεRACK peptide for 20 minutes, followed by 40 minutesperfusion without peptide. During the 60 minutes following ischemia (20minutes of peptide reperfusion plus 40 minutes perfusion), the CPKactivity in the perfusate was analyzed. The results are shown in FIGS.3A-3B.

[0096]FIG. 3A shows the CPK release as a function of time followingischemia for hearts treated with ψεRACK (open circles) and for heartsleft untreated after ischemia (solid triangles). FIG. 3B is a bar graphshowing the total CPK release during the 60 minute perfusion period forthe peptide treated and untreated ex vivo hearts. The data shows thatsubsequent administration of ψεRACK peptide to tissue previously exposedto an ischemic or hypoxic condition is effective to reduce the cellulardamage. FIG. 3B shows there was a nearly 2-fold lower total CPK releasefor tissue treated with ψεRACK peptide.

[0097] C. Adminstration of ψεRACK Peptide In Vivo

[0098] In another study in support of the invention, the ability ofψεRACK peptide to protect tissue from damage due to an ischemic orhypoxic event was evaluated by administering the peptide in vivo. Asdetailed in Example 3, ψεRACK peptide (SEQ ID NO:2) or scrambled ψεRACKpeptide (SEQ ID NO:4) was administered to adult pigs during the first 10minutes of a 30 minutes ischemic insult. Five days later, the heartswere analyzed for tissue damage. The results are shown in FIGS. 4A-4C.

[0099]FIGS. 4A-4B are computer-generated photos of pig heart slicestaken from the pigs treated in vivo with ψεRACK (FIG. 4A) or withscrambled ψεRACK peptide as a control (FIG. 4B). The hearts were stainedwith a double-staining technique (Example 3) that allowed determinationof the area at risk for ischemic injury (area within the arrows, mainlyin the mid wall, in FIG. 4B) and infarcted area (white area in FIG. 4B).As seen in FIG. 4B, control hearts treated with scrambled ψεRACK peptidehave a large infarct area within the area at risk (borders shown witharrows). In contrast, pigs that received the ψεRACK peptide (FIG. 4A)have a significantly reduced infarct area. By measuring surface areasand the weights of regions and total tissue weight of areas at risk andinfarcted regions, it was determined that the control hearts had anaverage of 36.5±0.3% infarct of area at risk, whereas hearts treatedwith ψεRACK peptide had an average of 14.9±0.6% infarct of area at risk(p<0.005).

[0100]FIG. 4C is a bar graph showing the infarct area as a percent ofarea at risk, measured in grams of cardiac tissue. As seen, the percentinfarct was reduced by more than 2-fold for the animals treated withψRACK peptide. Accordingly, delivery of a ψεRACK peptide in vivo priorto or during an ischemic event is effective to reduce the percentage ofinfarct by at least 2-fold.

[0101] Blood samples and tissue samples of lung, liver, brain, gut,kidney, etc. were collected from the animals and analyzed at a pathologylab. All samples were normal and no inflammation or tissue abnormalitieswere observed.

[0102] In another study, left ventricurogram (LVG) was performed in pigs(n=5) at three time points: (1) before occlusion of left anteriordescending artery by balloon catheter (before occlusion); (2)immediately after reperfusion with 5 μM/10 mL of ψεRACK (post ψεRACK);and (3) before sacrifice five days later (5 days post), using 6 Fr. ofpig-tail catheter. LVG was recorded by two views, right anterior obliqueand left anterior oblique. Ejection fraction (EF), the percent of bloodejected in a beat, during maximum contraction, of the total maximumpresent in the left ventricle, was analyzed by the software, Plus Plus(Sanders Data Systems), and the averages of two views were evaluated.Ejection fractions were calculated based on left ventricle dimensionsand the results are shown in FIG. 5. Ejection fraction is a measure ofhow well the heart is functioning, with a higher ejection fractionindicative of a better functioning heart. An ejection fraction of lessthan 50% in a short period of time can suggest progression into a stateof heart failure. As seen in FIG. 5, animals treated with ψεRACK (closedtriangles) had a higher ejection fraction after occlusion compared tothe control animals treated with a scrambled peptide (open circles),suggesting the peptide reduces or prevents damage to the cells andtissue due to ischemia. This is also evident from the data point at 5days post ischemia and treatment, where animals treated with ψεRACK hadan ejection fraction on par with that measured prior to ischemia andabout 10% higher than the untreated animals. Accordingly, delivery of aψεRACK peptide in vivo after ischemia is effective to reduce cell andtissue damage, as evidenced by an ejection fraction at least 10% greaterthan that of untreated cells or tissues.

[0103] III. ψεRACK Peptide

[0104] As used herein, a “ψεRACK peptide” refers to the peptiderepresented by SEQ ID NO:2 and to derivatives and fragments of thispeptide. Exemplary derivatives are given in SEQ ID NOS:6-18, and includethe following sequences: HEADIGYD (SEQ ID NO:6); HDAPIGYE (SEQ ID NO:7);HDAPVGYE (SEQ ID NO:8); HDAPLGYE (SEQ ID NO:9); HDAPIGDY (SEQ ID NO:10);HDAPIGEY (SEQ ID NO:11); ADAPIGYD (SEQ ID NO:12); HDGPIGYD (SEQ IDNO:13); HDAAIGYD (SEQ ID NO:14), and combinations of thesemodifications.

[0105] In one preferred embodiment, the sequence “DAPIG” (SEQ ID NO:18)in SEQ ID NO:2 is has no more than two modifications at any residue.One, two, or all three of the residues outside the sequence “DAPIG” canbe modified. For example, AEAPVGEY (SEQ ID NO:15) is a derivative of SEQID NO:2 where all three residues outside the “DAPIG” (SEQ ID NO:18)sequence and two residues within the “DAPIG” sequence are modified.Other examples include HEAPIGDN (SEQ ID NO:16) and HDGDIGYD (SEQ IDNO:17).

[0106] It will also be appreciated that fragments of SEQ ID NO:2 and ofthe modifications described above may be suitable. An exemplary fragmentof SEQ ID NO:2 is DAPIG, (SEQ ID NO:18).

[0107] All of these exemplary peptides may be (i) chemically synthesizedor (ii) recombinantly produced in a host cell using, e.g., an expressionvector containing a polynucleotide fragment encoding said peptide, wherethe polynucleotide fragment is operably linked to a promoter capable ofexpressing mRNA from the fragment in the host cell.

[0108] The dose of peptide administered will vary depending on thetissue to be treated and the condition of the patient. Dosages arereadily determined by those of skill in the art based on animal andhuman studies. Typically, between 0.05-5 μM, more preferably between0.1-2 μM, most preferably between about 0.1-1 μM peptide isadministered. However, the upper and lower limits of these ranges aremerely exemplary.

[0109] The peptide can be administered by any route suitable, asdetermine by the primary care provider. For example, administration byintraveneous, parenteral, subcutaneous, inhalation, intranasal,sublingual, mucosal, and transdermal, and the like, is contemplated.Naturally, the route of administration will influence the dose andtiming of administration, as appreciated by those of skill.

[0110] The peptide can be administered in the form of a fusion proteinor a transport protein conjugate. Typically, to form a fusion protein,the peptide is bound to another peptide by a bond other than a Cys-Cysbond. An amide bond from the C-terminal of one peptide to the N-terminalof the other is exemplary of a bond in a fusion protein. The secondpeptide to which the δPKC agonist/antagonist peptide is bound can bevirtually any peptide selected for therapeutic purposes or for transportpurposes. For example, it maybe desirable to link the ψεRACK peptide toa cytokine or other peptide that elicites a biological response.

[0111] Where the peptide is part of a conjugate, the peptide istypically conjugated to a carrier peptide, such as Tat-derived transportpolypeptide (Vives et al., 1997), polyarginine (Mitchell et al., 2000;Rolhbard et al, 2000) or Antennapedia peptide by a Cys-Cys bond. SeeU.S. Pat. No. 5,804,604. In another general embodiment, the peptide canbe introduced to a cell, tissue or whole organ using a carrier orencapsulant, such as a liposome in liposome-mediated delivery.

[0112] It will also be appreciated that ψεRACK as well as any compoundhaving similar activity can be used in the methods of treatmentdescribed herein. Other compounds, such as peptide mimetics, chemicalcompounds, or other peptides, can be identified by, for example, ascreening method set forth in U.S. Pat. No. 6,165,977, and this portionon Col. 14, line 45-Col 15, line 54 is incorporated by reference herein.In brief, and by way of example for identifying a compound effective toprotect a cell or tissue from ischemia, δPKC is immobilized inside thewells of a multiwell plate by introducing a solution containing δPKCinto the plate and allowing the δPKC to bind to the plastic. The wellsmay be precoated with substances that enhance attachment of δPKC and/orthat decrease the level of non-specific binding.

[0113] The plate is then incubated with a blocking solution (containing,for example bovine serum albumin) and then washed several times. Asolution containing reporter-labelled (e.g., radiolabelled offluorescently-tagged) peptide ψεRACK (SEQ ID NO:2) and, in the testwells, as opposed to the control wells, a test compound is added.Different wells may contain different test compounds or differentconcentrations of the same test compound. Each test compound at eachconcentration is typically run in duplicate and each assay is typicallyrun with negative (wells with no test compound) as well as positive(wells where the “test compound” is unlabeled peptide) controls. Thefree peptide is then washed out, and the degree of binding in the wellsis assessed.

[0114] A test compound is identified as active it if decreases thebinding of the peptide, i.e., if its effect on the extend of binding isabove a threshold level. More specifically, if the decrease in bindingis a several-fold different between the control and experimentalsamples, the compound would be considered as having binding activity.Typically, a 2-fold or 4-fold threshold difference in binding betweenthe test and control samples is sought.

[0115] Detection methods useful in such assays include antibody-basedmethods, direct detection of a reporter moiety incorporated into thepeptide, such as a fluorescent label, and the like.

[0116] A variety of test compounds may be screened, including otherpeptides, macromolecules, small molecules, chemical and/or biologicalmixtures, fungal extracts, bacterial extracts or algal extracts. Thecompounds can be biological or synthetic in origin.

[0117] IV. Utility and Routes of Administration

[0118] The present invention has application, for example, in treatmentof surviving heart attack victims, as well in treatment of persons whopresently die from heart disease after admission to the hospital.Delivery of the εPKC selective peptide agonist, ψεRACK, is valuable inthe management of these patients, both acutely and chronically.

[0119] Acutely, in patients brought to hospital with impendinginfarction, medical care has traditionally been directed towardsremoving the cause of coronary occlusion either by thrombolytics or bycatheter angioplasty. However, reperfusion of the damaged areas can beone of the major mechanisms of myocardial cellular injury.Administration of a ψεRACK peptide PKC agonist delivered to the site ofocclusion by catheter or injected intravenously to inducecardioprotection immediately before or concurrently with thrombolysis orangioplasty is contemplated by the invention.

[0120] Chronically, in patients with angina, the current medicalapproach is to stop the symptoms of angina without replacement ofangina's preconditioning protective effect. AεPKC selective agonist,such as ψεRACK, can replace the preconditioning effect induced by anginain these patients and offer a higher rate of myocardial salvage duringfuture episodes of more severe ischemia.

[0121] Additional uses of the invention include clinical situations inwhich the timing of ischemia is physician-controlled. In such instances,pharmacologic enhancement of the preconditioning response would providea significant advantage to the patients undergoing treatment.Specifically, each year, in the United States alone, 600,000 adults and12,000 children undergo open heart operations utilizing cardiopulmonarybypass, during which the heart is subjected to periods of controlledischemia ranging from several minutes to well over one hour. Despiteadvances in cardiac protection, myocardial dysfunction during theimmediate post-operative period remains a leading cause of morbidity andmortality in these patients. The exact timing of the ischemic insult isknown ahead of time in these patients, allowing for administration of aψεRACK peptide prior to ischemia. Administration of ψεRACK will reducemyocardial damage by inducing a preconditioning response in the hours,or days, prior to surgery.

[0122] Similar benefits could be realized in the area of cardiactransplantation, of which there are approximately 2500 cases annually inthe U.S. Prolonged graft ischemia is one of the factors limitinglong-distance donor organ acquisition for such cardiac transplantation.Administration of ψεRACK peptide at the time of organ procurement couldextend the time between organ harvest and implantation and reduce therisk of post-operative myocardial dysfunction.

[0123] It will, of course, be understood that a ψεRACK peptide mayemployed in the treatment of a variety of ischemic and hypoxicconditions, in addition to cardiac ischemia. For example, ψεRACK may beadministered prior to, during or after an ischemic or hypoxic event to awide variety of cells and tissues. Without intending to be limiting,examples include the kidney, the vascular endothelium, the liver, theeye, and in the central nervous system, where tissue damage to the brainand other tissues of the central nervous system may result due tostroke.

[0124] As demonstrated by the studies described herein, the peptide canbe administered before, during or after an ischemic or hypoxic event.When delivered before an ischemic insult, the peptide effectivelyreduces the extent of cellular damage. Preferably, the peptide isperfused over or through the tissue for about 1-180 minutes, morepreferably for about 1-120 minutes, most preferably for about 1-60minutes, prior to the ischemic insult. In one embodiment, a period oftime not greater than about three hours, more preferably not greaterthan about 120 minutes, and most preferably not greater than about 60minutes, lapses between cessation of peptide perfusion and the ischemicor hypoxic event. The peptide can be delivered for a 1 minute, 2 minute,5 minute, 10 minute, 20 minute, 30 minute, or longer, period of timeprior to the ischemic insult.

[0125] When the peptide is delivered subsequent to an ischemic event, ina preferred embodiment a period of time not greater than about twohours, more preferably not greater than one hour, and even morepreferably not greater than 30 minutes lapses between the ischemic eventand initiation of administration of the peptide. The peptide can bedelivered for a 1 minute, 2 minute, 5 minute, 10 minute, 20 minute, 30minute, or longer, period of time following the ischemic insult.

[0126] The peptide can also be administered during an ischemic event.Particularly, during time of controlled ischemia, such as duringsurgery, the care provider can initiate administration of the ψεRACKpeptide just prior to or concurrent with initiation of the ischemicevent.

V. EXAMPLES

[0127] The following examples further illustrate the invention describedherein and are in no way intended to limit the scope of the invention.

[0128] Methods

[0129] 1. Peptide Preparation

[0130] ψεRACK (HDAPIGYD, SEQ ID NO:2) was synthesized and purified(>95%) at the Stanford Protein and Nucleic Acid Facility. ScrambledψεRACK peptide (PDYHDAGI, SEQ ID NO:4) was prepared similarly. In somestudies, the peptides were modified with a carrier peptide bycross-linking via an N-terminal Cys-Cys bond to the DrosophilaAntennapedia homeodomain-derived peptide (C-RQIKIWFQNRRMKWKK, SEQ IDNO:3; Theodore, L. et al., 1995; Johnson, J. A. et al., 1996a) or via anN-terminal Cys-Cys bond to Tat protein-derived peptide (C-YGRKKRRQRRR,SEQ ID NO:6).

Example 1 Ex Vivo Administration of ψεRACK Prior to Ischemia

[0131] Mice or rats were anesthetized with i.p. avertin, and theirhearts were rapidly removed and cannulated via the aorta for reperfusionas described in the art (Colbert et al, 1997). Care was taken to havethe hearts perfused within 90 seconds of removal. The left ventricularpressure and real-time derivative (dP/dt) were monitored via a catheterplaced in the ventricular apex. Hemodynamic parameters were archivedevery 20 seconds throughout the procedure. The hearts were perfused withoxygenated Krebs-Henseleit solution comprised of, in nmol/L, NaCl 120;KCl 5.8; NaHCO₃ 25; NaH₂O₄ 1.2; MgSO₄ 1.2; CaCl₂ 1.0; and dextrose 10,pH 7.4 at 37 C.

[0132] After a 10-20 minute equilibration period, the hearts weretreated with ψεRACK peptide (SEQ ID NO:2) or with scrambled ψεRACKpeptide (SEQ ID NO:4) for 20 minutes. Perfusion was maintained at aconstant flow of 10 mL/min with Krebs-Henseleit solution containing 0.5εM of the appropriate peptide. The Langendorff method employed usedretrograde flow from the ventricle to the aorta and into the coronaryarteries, bypassing the pulmonary arteries.

[0133] To induce global ischemia, flow was interrupted for 45 minutes.After the ischemic event, the hearts were reperfused withKrebs-Henseleit solution for 30-160 minutes. During reperfusion,ischemia-induced cell damage was determined by measuring the activity ofcreatine phosphokinase (CPK) (absorbance at 520 nm) in the perfusateusing a Sigma kit. The results are shown in FIGS. 1A-1B.

Example 2 Ex Vivo Administration of ψεPKC After to Ischemia

[0134] Rat hearts were prepared as described in Example 1. After a 30minute equilibration period, global ischemia was induced by interruptingfluid flow for 45 minutes. The hearts were then reperfused with 0.5 μMof ψεRACK peptide for 20 minutes, followed by 40 minutes of reperfusionwithout the peptide. As a control, some hearts were left untreated afterischemia. During the 60 minute period following ischemia,ischemia-induced cell damage was determined by monitoring the creatinephosphokinase (CPK) activity (absorbance at 520 nm) in the perfusatecollected during reperfusion. The results are shown in FIGS. 3A-3B.

Example 3 In Vivo Administration of ψεPKC Prior to Ischemia

[0135] Young adult female pigs, 35-40 kg in weight, were anesthetizedand a catheter was introduced through the carotid artery into the heart.Using conventional intervention cardiology techniques, a wire was placedthrough a catheter and into the left anterior descending artery (LAD). Aballoon was run over this wire to a site of occlusion where it was theninflated to block blood flow for 30 minutes. During the first 10 minutesof the ischemic insult, either the control scrambled ψεRACK peptide (SEQID NO:4, n=5) or the biologically active ψεRACK peptide (SEQ ID NO:2,n=5) was delivered by diffusion through the balloon directly downstreamof the occlusion. Approximately 20 μg peptide (400 ng per kg bodyweight) was administered.

[0136] After 30 minutes of occlusion, the balloon was removed and pigswere left to recover from surgery. Five days later, the pigs wereeuthanized and hearts were harvested. After heart removal, the LAD wasoccluded. With the occlusion in place, Evans Blue dye, which stains allareas not at risk of infarct in blue while leaving all areas with noaccess to blood flow red, was infused. Hearts were then cut into slicesand stained with a tetrazolium red dye which stains all live areas redand infarcted dead tissue in white. Each heart had multiple tissueslices with distinctive areas marking the area at risk for ischemia andthe infarcted area. From this the percent infarct per area at risk foreach slice and for the entire heart was determined. The results areshown in FIGS. 4A-4C.

[0137] Although the invention has been described with respect toparticular embodiments, it will be apparent to those skilled in the artthat various changes and modifications can be made without departingfrom the invention.

1 18 1 8 PRT Artificial Sequence epsilon V1-2, residues 14-21 ofepsilon-PKC 1 Glu Ala Val Ser Leu Lys Pro Thr 1 5 2 8 PRT ArtificialSequence pseudo-epsilon RACK octapeptide 2 His Asp Ala Pro Ile Gly TyrAsp 1 5 3 16 PRT Artificial Sequence Drosophila antennapediahomeodomain-derived carrier peptide 3 Arg Gln Ile Lys Ile Trp Phe GlnAsn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 4 8 PRT Artificial Sequencescrambled pseudo-epsilon RACK octapeptide 4 Pro Asp Tyr His Asp Ala GlyIle 1 5 5 11 PRT Artificial Sequence Tat-derived carrier peptide 5 TyrGly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 6 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 6 His Glu Ala Asp Ile GlyTyr Asp 1 5 7 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 7 His Asp Ala Pro Ile Gly Tyr Glu 1 5 8 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 8 His Asp Ala Pro Val GlyTyr Glu 1 5 9 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 9 His Asp Ala Pro Leu Gly Tyr Glu 1 5 10 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 10 His Asp Ala Pro Ile GlyAsp Tyr 1 5 11 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 11 His Asp Ala Pro Ile Gly Glu Tyr 1 5 12 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 12 Ala Asp Ala Pro Ile GlyTyr Asp 1 5 13 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 13 His Asp Gly Pro Ile Gly Tyr Asp 1 5 14 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 14 His Asp Ala Ala Ile GlyTyr Asp 1 5 15 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 15 Ala Glu Ala Pro Val Gly Glu Tyr 1 5 16 8 PRT ArtificialSequence modified pseudo-epsilon RACK peptide 16 His Glu Ala Pro Ile GlyAsp Asn 1 5 17 8 PRT Artificial Sequence modified pseudo-epsilon RACKpeptide 17 His Asp Gly Asp Ile Gly Tyr Asp 1 5 18 5 PRT ArtificialSequence pseudo-epsilon RACK peptide fragment 18 Asp Ala Pro Ile Gly 1 5

It is claimed:
 1. A method of reducing injury to a cell exposed to anischemic or an hypoxic condition, comprising administering to the cell aψεRACK peptide.
 2. The method of claim 1, wherein said administeringoccurs prior to exposing the cell to said ischemic or hypoxic condition.3. The method of claim 2, wherein said administering prior to saidischemic or hypoxic condition is for a period of time of between about1-180 minutes prior to said exposing.
 4. The method of claim 1, whereinsaid administering occurs after exposing the cell to said ischemic orhypoxic condition.
 5. The method of claim 4, wherein said administeringafter exposure to said ischemic or hypoxic condition occurs for betweenabout 1-180 minutes after said ischemic or hypoxic condition.
 6. Themethod of claim 1, wherein said administering occurs during exposure ofthe cell to said ischemic or hypoxic condition.
 7. The method of claim 1wherein said administering includes administering a peptide having asequence identified as SEQ ID NO:2.
 8. The method of claim 1, whereinsaid administering includes administering a peptide having a sequenceselected from the group consisting of SEQ ID NOS:6-18.
 9. The method ofclaim 1, wherein said administering includes administering a ψεRACKpeptide linked to a moiety effective to facilitate transport across acell membrane.
 10. The method of claim 9, wherein the moiety is selectedfrom the group consisting of a Tat-derived peptide (SEQ ID NO:5), anAntennapedia carrier peptide (SEQ ID NO:3), and a polyarginine peptide.11. The method of claim 1, wherein said administering includesadministering the peptide by a route selected from the group consistingor intraveneous, parenteral, subcutaneous, inhalation, intranasal,sublingual, mucosal, and transdermal.
 12. A method of reducing injury totissue exposed to an ischemic or an hypoxic condition, comprisingadministering to the tissue a ψεRACK peptide.
 13. The method of claim12, wherein said administering occurs prior to exposing the tissue tosaid ischemic or hypoxic condition.
 14. The method of claim 13, whereinsaid administering prior to said ischemic or hypoxic condition is forbetween about 1-180 minutes.
 15. The method of claim 12, wherein saidadministering occurs after exposing the tissue to said ischemic orhypoxic condition.
 16. The method of claim 15, wherein saidadministering after exposure to said ischemic or hypoxic conditionoccurs for between about 1-180 minutes after said ischemic or hypoxiccondition.
 17. The method of claim 12, wherein said administering occursduring exposure of the tissue to said ischemic or hypoxic condition. 18.The method of claim 12, wherein said administering includesadministering a peptide having a sequence identified as SEQ ID NO:2. 19.The method of claim 12, wherein said administering includesadministering a peptide having a sequence selected from the groupconsisting of SEQ ID NOS:6-18.
 20. The method of claim 12, wherein saidadministering includes administering a ψεRACK peptide linked to a moietyeffective to facilitate transport across a cell membrane.
 21. The methodof claim 12, wherein the moiety is selected from the group consisting ofa Tat-derived peptide (SEQ ID NO:5), an Antennapedia carrier peptide(SEQ ID NO:3), and a polyarginine peptide.
 22. The method of claim 12,wherein said administering includes administering the peptide by a routeselected from the group consisting or intraveneous, parenteral,subcutaneous, inhalation, intranasal, sublingual, mucosal, andtransdermal.
 23. The method of claim 12 wherein said administering is toa tissue that is a whole organ ex vivo.
 24. The method of claim 12wherein said administering is to a tissue that is a whole organ in vivo.25. The method of claim 23 or 24, wherein said organ is selected fromthe group consisting of heart, lung, liver, brain, and kidney.
 26. Themethod of claim 24, wherein said administering is by infusion throughcoronary arteries to an intact heart.